Storage-stable fibrin sealant

ABSTRACT

Provided are supplemented and unsupplemented, ready-to-use and instantly-available fibrin sealants (“FS”), prepared from ready-to-use, storage-stable, concentrated liquid fibrinogen preparations. The thus-produced FS product when applied to a tissue provides the elasticity, tensile strength, and adhesiveness necessary to prevent blood loss, to promote wound healing, and for many other therapeutic and non-therapeutic applications. Further provided are kits for, and methods of preparation of, the supplemented and unsupplemented, storage-stable FS products of the present invention, and methods of use and delivery therefor.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to supplemented and unsupplemented,storage-stable fibrin sealants. More specifically, the subject inventionrelates to instantly-available fibrin sealants prepared fromstorage-stable, ready-to-use concentrated fibrinogen preparations, tomethods of preparing such fibrin sealants, and to methods of usetherefor to prevent blood loss, to promote wound healing, and for manyother therapeutic and non-therapeutic applications.

Clot formation in humans, i.e., blood coagulation, occurs by means of acomplex cascade of events in which in the final steps the monomeric formof fibrinogen reacts with thrombin and activated Factor XIII in thepresence of calcium ions, to form a fibrin clot comprising across-linked fibrin polymer. Recently, biological adhesives have beendeveloped comprising fibrinogen, thrombin and one or more othercomponents, which imitate the final stages of natural coagulation,thereby resulting in a fibrin clot. Thus, the fibrinogen-based material,known as fibrin- or tissue-sealant, biological sealant, fibrin- ortissue-glue, biological adhesive, surgical adhesive, or the like(collectively referred to herein as a “fibrin sealant,” or “FS”), can beused to join living tissue together, and keep it joined to seal internaland external wounds, e.g., in tissue, organs, muscle, bones and skin,and to reduce blood loss while producing a hemostatic action (see forexample patent FR-2448900). Such adhesives are commonly used in surgery,particularly to prevent or stop bleeding, replace or reinforce suturethreads, hold grafts in place, e.g., skin grafts, to seal resectionedtissues, e.g., in lung or gastrointestinal tract surgery, or to glueparts of prostheses, etc.

FS products generally are prepared from: (1) fibrinogen concentrate,which may also contain fibronectin, Factor XIII, von Willebrand factorand traces of albumin; (2) an activator component such as thrombin(e.g., human or bovine) or a thrombin-like material; and (3) a thrombinactivator, such as calcium ions (e.g., CaCl₂). The precise compositionof each ES, however, is a function of the particular plasma fraction(s)used as the starting material. For example, commercially prepared FSproducts often contain bovine components. In Canada, Europe, andpossibly elsewhere, commercially available FS typically also containsaprotinin as a stabilizer. Nevertheless, a direct relationship has beenshown between tensile strength and the final fibrinogen concentration(Japanese Patent Unexamined Published Application, Kokai No. Sho61-293443). Thus, the availability of concentrated fibrinogen is animportant factor for the preparation of conventional FS products.

Australian Patent 75097/87 describes a one-component adhesive, whichcontains an aqueous solution of fibrinogen, Factor XIII, a thrombininhibitor, such as antithrombin III, prothrombin factors, calcium ions,and, if necessary, a plasmin inhibitor. U.S. Pat. Nos. 4,427,650 and4,427,651 (Stroetmann), describe the preparation of an enriched plasmaderivative in the form of a powder or sprayable preparation for enhancedwound closure and healing that contains fibrinogen, thrombin and/orprothrombin, and a fibrinolysis inhibitor, and may also contain otheringredients, such as a platelet extract. U.S. Pat. Nos. 4,627,879 and4,928,603 (Rose et al.), disclose methods for preparing cryoprecipitatedsuspensions that contain fibrinogen and Factor XIII and their use toprepare a FS. JP 1-99565 discloses a kit for the preparation of fibrinadhesives for wound healing.

PCT document WO91/09641 describes a fibrin glue containing fibrinogenand added thrombin. This FS contains added thrombin, but is prepared insuch a way that the thrombin activity is inhibited, and in oneembodiment it comprises no calcium ions, as these are not added untilthe time of use. However, the disclosed FS tends to coagulatespontaneously after about 90 seconds, even without the addition ofcalcium ions. When calcium ions are added, it coagulates in less than 2seconds. In other embodiments, coagulation of the glue is slowed down byacidifying the product to a pH of less than 5.5 to inhibit thrombinactivity. The disclosure further provides a means of increasing the pHat the time of use to nullify the inhibition effect,

In addition, FS delivery systems have been disclosed by Miller et al.,U.S. Pat. No. 4,932,942 and Morse et al., PCT publication WO 91/09641.FS products have been commercially marketed for a number of years inEurope by Immuno AG (Vienna, Austria) and Behringwerke AG (Germany)(Gibble et al., Transfusion 30:741-747 (1990)) and elsewhere (see, e.g.,U.S. Pat. Nos. 4,377,572 and 4,298,598, owned by Immuno AG).

However, most FS products used clinically outside of the U.S. posecertain risks and, as a result have not been approved by the Food andDrug Administration for use in the U.S.A. For example, as noted above,the FS products available in Europe contain proteins of non-humanorigin, e.g., aprotinin and bovine thrombin. Consequently, certainindividuals are at risk of developing allergic reactions to suchnon-human protein additives. U.S. Pat. No. 6,183,498 reports that theuse of biomedical adhesives have been observed to induce inflammatorytissue reactions.

Moreover, when heat inactivation is used to inactivate any viruses thatmay be present in the FS, the process may result in the formation ofdenatured proteins, which may also be allergenic. For example, theEuropean heat inactivation methods do not inactivate prions which causebovine spongiform encephalopathy (“mad cow disease”), which has beenepidemic recently in bovine herds in European, and. hence disease couldbe carried in the bovine proteins used in the foreign FS products,risking human infection when those products are used for their intendedpurpose.

Alterbaum (U.S. Pat. No. 4,714,457) and Morse et al. (U.S. Pat. No.5,030,215) disclose methods to produce autologous FS in which no bovineproducts are used. PCT publication WO94/07548 describes FS enriched withplatelet factors that is able to coagulate without addition of thrombinby adding to the recalcified glue, a coagulation activator, such askaolin. However, because the activator is not incorporated until thetime the glue is used, the time coagulation time is uncertain anddifficult to predict or control. This is because the fibrinogenconcentrate is a highly viscous product, which is difficult to handle.Moreover, since coagulation progresses simultaneously with activation,it is difficult to separate the activator from time activated glue.

Nevertheless, at a sufficiently high fibrinogen concentration, FSpreparations provide safe hemostasis, good adherence of the seal to thewound and/or tissue areas, high strength of the adhesions and/or woundsealings, and complete resorbability of the adhesive in the course ofthe wound healing process (Byrne et al., Br. J. Surg. 78:841-843(1991)). For optimal adhesion, a concentration of fibrinogen of about 15to 60 mg/ml is required in a ready-to-use tissue adhesive solution(MacPhee, personal communication). The clinical uses of FS products havebeen reviewed (e.g., by Brennan, Blood Reviews 5:240-244(1991); Gibbleet al., Transfusion 30:741-747 (1990); Matras, J. Oral Maxillofac. Surg.43:605-611 (1985); Lerner et al, J. Surg. Res. 48:165-181 (1990)).

Baxter/Hyland (Los Angeles, Calif.) in conjunction with The AmericanNational Red Cross have co-developed Tisseel, the first commercialfibrin sealant to be approved in the United States (see, e.g., U.S. Pat.Nos. 6,054,122; 6,117,425; and 6,197,325 (MacPhee et al.)). This FSproduct has advantages over those available in Europe because it is freeof bovine proteins. For example, it contains human thrombin, and itcontains no aprotinin, thereby reducing the potential for allergenicity.In addition, it is virally inactivated by a solvent detergent method,which produces fewer allergenic denatured proteins.

From the standpoint of preparation, the fibrinogen component of the FScan be prepared from plasma by cryoprecipitation, followed byfractionation, to yield a composition that forms a fibrin sealant, orclot, upon exposure to, or mixing with activated thrombin. In the priorart, the fibrinogen and thrombin concentrates are stored in lyophilizedform that must be reconstituted and mixed with a solution of CaCl₂immediately prior to use. Upon mixing, the components are applied to atissue where they coagulate on the tissue surface and form across-linked fibrin clot, Factor XIII, which is present in thefibrinogen concentrate, catalyzes the cross-linking.

According to U.S. Pat. No. 5,290,552, early surgical adhesiveformulations necessarily contained a high fibrinogen content (about8-10%), from which lyophilates were extremely difficult to prepare. Infact, cryoprecipitates of concentrated fibrinogen are known to be highlyunstable in liquid solution, thus requiring storage below −20° C. untiluse (http:www.tissuesealing.com/us/products/biological/monograph.cfm);i.e., in aqueous form concentrated fibrinogen is subject to spontaneouscoagulation. Consequently, commercially available lyophilized and/ordeep-frozen fibrinogen concentrates, such as Tissucol, must beliquefied, i.e., slowly thawed (“melted”) or reconstituted fromlyophilates before application. Both liquefaction processes, however,are associated with significant effort and a considerable time lagbefore time product can be used in FS products, which can place analready injured patient into a life-threatening situation.

Therefore, significant effort has been undertaken to improve thesolubility of lyophilized fibrinogen preparations. For example, onemanufacturer requires the use of a magnetic stirrer added to the vialsof protein to provide significant agitation while heating. This resultsin dissolution times which are faster than those obtained for the sameproduct without significant mixing, but it still requires 30-60 minutesof preparation time simply to get the fibrinogen ready to use.

U.S. Pat. No. 5,962,405 provides storage-stable lyophilized or deepfrozen liquid preparations of fibrinogen, which can be reconstituted andliquefied into ready-to-use fibrinogen and/or tissue adhesivesolutions—preferably without the use of additional means, such asheating and/or stirring devices, to produce ready-to-use tissue adhesivesolutions having a fibrinogen concentration of at least 70 mg/ml. Thepreparations comprise fibrinogen and at least one additional substancewhich improves the solubility of the preparations, and/or lowers itsliquefaction temperature, and reduces the viscosity of a ready-to-usetissue adhesive solution at room temperature. However, because theliquefaction temperature is lowered, the '405 patent claims thatliquefaction of the deep-frozen, concentrated fibrinogen solution isadvantageously possible in a surrounding temperature of 20° to 23° C.(room temperature), as opposed to the previously required 37° C. warmingconditions. Nevertheless, the method still requires storage underdeep-frozen conditions (temperatures maintained at −15° C. to below −25°C.), and the preparations still take up to 15 minutes to liquefy.

Instructions for the previously mentioned Tisseel fibrin sealant(Baxter) indicate that preparation of the fibrinogen and thrombincomponents takes at least 15 minutes. The Baxter sealer proteinconcentrate (fibrinogen) is provided as a. freeze-dried powder, which isreconstituted by mixing with a fibrinolysis inhibitor solution. TheBaxter thrombin component, which also comes as a freeze-driedconcentrate, is reconstituted using a calcium chloride solution.Preparation of each component in the Baxter kit can be semi-automatedusing the optionally provided fibrinotherm heating and stirring device.To accommodate the protein preparation processes, each sealer proteinconcentrate vial contains a magnetic stir bar that fits into acustom-sized stirring well for uniform mixing at optimal physiologictemperature (37° C.).

However, not only does the need to slowly liquefy the protein componentscause a significant delay in the formation of the FS preparation, asignificant problem arises once fibrinogen is solubilized because itsinstability results in a tendency to prematurely self-coagulate. Infact, once prepared, the Baxter instructions indicate that thereconstituted solutions can be kept in their respective vials orsyringes for a maximum of only 4 hours, after which any unused sealantmust be discarded. As a result, the Baxter FS cannot be stored in aready-to-use condition for any useful length of time.

As one solution to overcome the need to reconstitute or liquefy thelyophilized or deep-frozen fibrinogen products before use, especiallyconcentrated preparations, certain fibrinogen preparations have beenintroduced which are soluble at room temperature. Unfortunately,however, such prior art products have proven to be cytotoxic (Beriplast,Biocol, Bolheal HG-4).

In an alternative solution, to delay the tendency of fibrinogen inaqueous solution to prematurely coagulate, U.S. Pat. No. 5,985,315provides a stable biological pre-activated adhesive comprisingfibrinogen with the addition of at least one activated coagulationfactor whose activation does not depend on calcium ions. Thepreactivated adhesive is stable in aqueous solution, i.e., the solutiondoes not coagulate spontaneously for at least one hour at a temperatureof 20° C.; but it can be made to coagulate in about 5 minutes simply byadding calcium ions. No additional activator is required. Thus, theresulting biological adhesive requires neither the addition of thrombinor prothrombin to achieve coagulation. Unfortunately, however, 5 minutesis a very slow coagulation time, making the use of the resulting fibrinsealant impractical for use on any type of a flowing or pulsating wound,e.g., anastomoses, blood vessels, airholes in lung injuries, or injuriesto parenchymal or bronchiole tissue.

From a medical standpoint therefore, the quick availability ofready-to-use, biological, tissue adhesives is essential, especially insurgical emergency situations. Despite continued advances in traumacare, a significant percentage of the population, both military andcivilian, suffer fatal or severe hemorrhage every year. An alarmingnumber of fatalities are preventable since they occur in the presence ofthose who could have achieved lifesaving control of their wounds, givenadequate tools and training. Thus, there is a recognized need for anadvanced, easy-to-use, field-ready hemostatic preparation, permittingnot only trained medical personnel, but even untrained individuals torapidly reduce bleeding in trauma victims. The effect of this need istwo-fold: a significant number of trauma deaths could be prevented, andthe demand upon the available blood supply could be reduced.

When presented with a large number of victims from severe natural orman-made disasters, local hospitals and clinics may be overwhelmed bythe number of individuals requiring trauma care. Often the resultingdemand for blood and blood products exceeds the locally availablesupplies; and in many cases, the demand for assistance exceeds theavailability of trained medical personnel. However, the availability ofa ready-to-use, self-contained FS preparation would permit local medicalpersonnel and disaster relief workers to provide the injured withtemporary treatment until definitive care becomes available. Suchready-to-use, storage-stable FS preparation(s) would become a valuabletool for emergency care providers, and on ambulances and rescuevehicles. As a result ready-to-use, storage-stable FS preparations willallow anyone to teat an injury victim, or even permit self-treatment,until medical assistance can be provided, making such a FS a valuablecomponent of first-aid kits for the home, car, or office or on publictransportation.

Ideally, the FS product should require as little manipulation aspossible in its preparation, to minimize risks and the burden on theassisting personnel. Currently, fibrinogen-based FS preparations requirea fibrinogen component that is available only as a lyophilate, adeep-frozen concentrate, or as a mixture with other components thatcould negatively alter hemostasis, or its safe use with a human patient.Thus, until the present invention there has remained a. need for aready-to-use FS composition that is rapidly prepared from astorage-stable, aqueous fibrinogen solution, which despite its highconcentration, remains available in fluid form, permitting easyprocessing into the instantly-available FS product for use on humans oranimals, and which is both safe and effective, without risk of adverseeffects.

SUMMARY OF THE INVENTION

The present invention provides supplemented and unsupplemented,ready-to-use and instantly-available fibrin sealants (“FS”), preparedfrom ready-to-use, storage-stable, concentrated liquid fibrinogenpreparations. The thus-produced FS product when applied to a tissueprovides the elasticity, tensile strength, and adhesiveness necessary toprevent blood loss, to promote wound healing, and for many othertherapeutic and non-therapeutic applications. It further providesmethods of preparation of the supplemented and unsupplemented,storage-stable FS products of the present invention, and methods of usetherefor.

The supplemented and unsupplemented, storage-stable FS products of thepresent invention are unique in that they are advantageously instantlyavailable in ready-to-use form because the components used in theirpreparation are storage-stable and ready-to-use. Specifically, thefibrinogen component of the present FS is biocompatible, and remainsavailable in fluid form at appropriate concentrations to permit rapidand easy preparation of FS. The sterile, storage-stable fibrinogen isaqueous and fully solubilized, its stability is pH and temperaturedependent, and it retains its biological activity (i.e., the ability torapidly form a fibrin clot upon exposure and vigorous mixing withthrombin and calcium ions). The thus prepared and stored, ready-to-use,concentrated human fibrinogen solutions may be neutralized and usedwithout additional steps or processes in the preparation ofbiocompatible instantly available FS compositions.

One of the benefits of fibrin sealants is the natural bioabsorption thatoccurs after the cross-linked fibrin product has sealed the wound. Thisaction, resulting from plasmin mediated lysis, permits natural removalof the fibrin sealant from the body and provides methods for acceleratedremoval if needed. This property, along with the superior adhesivenessand elasticity of the FS product, contributes to the value andversatility of the FS products as an emergency treatment that can beprocessed by hospital personnel once the patient is received or as anadjunct to surgical procedures. The FS product may advantageously beused directly on open wounds, or its use may be combined with otherbandaging or suturing systems.

It is therefore an object of the present invention to provide aready-to-use FS composition which can rapidly form a strong, yetflexible biologically compatible bond between separated tissues, or toachieve a coating or seal of a wound or undesirable opening in a tissue,to apply a graft, to coat a prosthetic material, or to deliver additivecompounds to the surrounding tissue or circulatory systems. It ispreferred that the resulting bond, covering or seal be watertight. Suchcomposition is effective for its intended purpose on tissue in vitro, aswell as in vivo in a human or animal patient. Further it is an object ofthe invention to provide FS compositions in which viscosity and/orpolymerization time can be modified according to the desired applicationto facilitate placement of the composition at the tissue site.

It is still another object of the invention to provide a method ofbonding separated tissues, of sealing or coating a wound or tissues toform a watertight seal, of applying a graft, or of coating a prostheticmaterial using a FS composition which is easy to handle, particularlyduring surgical procedures.

It is yet another object of the invention to provide methods offormulating such FS products, as well as methods of using same in vitaror in vivo to seal wounds, to apply grafts, to coat prostheses, or todeliver additive compounds to the surrounding tissue or circulatorysystems.

It is also an object of the invention to provide methods of applying theinstant FS components and the final FS product to the tissue or woundsite. A particular advantage of the invention is the ready-to-useavailability of the components, including the storage-stable fibrinogencomponent as an aqueous solution. Thus, the components are combinedeither immediately before application to the tissue or wound site orsimultaneously with application to form the FS product. Dual syringedelivery devices can be easily used with consistent results using thedescribed components of the instant FS composition because thecomponents are stored in ready-to-use condition, and may, in fact, beseparately and stabily stored within the barrels of the syringe for easeof delivery.

In the alternative, the components may be separately and stabily storedin divided compartments within a single barrel syringe, such that anaffirmative action, such as pressing the plunger will cause the barrierto open permitting the pre-measured components to mix. Delivery of theFS composition is then instantly directed from a single port or needleto the tissue or wound site allowing the polymerization andcross-linking of the fibrin clot to occur directly at the site. In yetanother alternative, syringe devices (single or multi-barrel) may beused to draw the storage-stable components from larger storagecontainers using standard syringe techniques, and the components or themixed FS composition is delivered as described above, so long as fibrinpolymerization and cross-linking occurs at the wound site.

It is a further object to provide kits for the ready-to-use delivery ofan instant FS composition comprising at least two vials. One vialcontains an aqueous solution of storage-stable fibrinogen at aconcentration suitable for forming FS when mixed with an activatorsolution, such as activated thrombin or a thrombin-like composition, anda second vial contains the activator solution (preferably thrombin) at aconcentration suitable for forming FS when mixed with the contents ofthe storage-stable fibrinogen in the first vial. CaCl₂ is added to andstored with the contents of one of the at least two vials in aneffective amount to ensure fibrin polymerization, or in the alternative,the CaCl₂ component is supplied in an additional vial. Additionalcomponents, such as a stabilizer and/or Factor XIII and/or additives,such as a growth factor, drag, antibiotic, and the like are supplied byone or more additional vials, or alternately such additional componentsare added to and stored with the contents of the at least two vials.

Notably, however, a vial of The kits herein provided is expresslyintended to also include a barrel of a syringe device. Accordingly, inone embodiment, the kit includes the described components provided in asingle divided barrel or multi-barrel syringe device, so long as thefibrinogen component and the activator component remain separated untilthe instant FS composition is mixed and delivered.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description, examples and figures whichfollow, and in part will become apparent to those skilled in the art onexamination of the following, or may be learned by practice of theinvention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention comprises supplemented and unsupplemented, storage-stablefibrin sealants (“FS”), prepared from ready-to-use, storage-stable,concentrated liquid fibrinogen solutions. The present FS is novelbecause it is instantly available since the components therefor arestorage-stable and ready-to-use. In particular, the fibrinogen componentis a shelf-ready aqueous solution, which is “storage-stable,” that is,after a period of days, weeks, months or longer, it remains stable inliquid form, it has not spontaneously clotted (i.e., it has not formed a“spontaneous clot,” even in the absence of an activator, such asthrombin/Ca⁺⁺), and it retains its biological activity (i.e., theability to rapidly form a fibrin clot upon exposure and vigorous mixingwith thrombin and Ca⁺⁺). The disclosed methods, therefore, set forth theconditions under which the FS components, including fibrinogen, isstored in a ready-to-use, aqueous solution for a period of days andremains active and stable (storage-stable).

The FS composition of the present invention is noninfectious, andprovides a tissue bond having high tensile strength, elasticity,deformability, water tightness, viscosity and adhesivity for a largevariety of surgical procedures. The composition can also be used to coatimplantable devices to enhance their strength and resistance to fluids,to seal pores in the weave of a material implant device, and to reducethrombogenicity. In the present disclosure, unless defined otherwise,all technical and scientific terms used herein have the same meaning asis commonly understood by one of ordinary skill in the art to which theinvention pertains.

The FS composition of the subject invention comprises a fibrin polymerprepared from any form of fibrin monomer. In a preferred embodiment ofthe present invention, the FS fibrin polymer forms “instantly” or withinseconds of activation of the storage-stable fibrinogen component of thepreparation.

The enzymatic conversion of fibrinogen to fibrin is a two-step process.First, activated thrombin or a thrombin-like molecule cleaves the outerA and B terminal peptides of the fibrinogen molecule to form a solublemonomer, fibrin I, that is susceptible to internal catalysis. ActivatedFactor XIII (which is up-regulated by the conversion of prothrombin tothrombin) catalyzes the formation of amide bonds between a pair of aminoacids in the fibrinogen monomers, resulting in the final cross-linked,insoluble fibrin II matrix. The cleavage only slightly reduces themolecular weight of fibrinogen from 340,000 daltons to only 334,000daltons, but the process exposes the essential polymerization sites topermit formation of the assembled and cross-linked fibrin clot. See,Jackson. Ann. Rev. Biochem. 49:765-811 (1980); Furie et al, Cell53:505-518 (1988).

The conversion of fibrinogen into fibrin via the enzymatic activities ofactivated thrombin and Factor XIII occurs under precise physiologicconditions. Exogenous generation of a matrix that possesses the superiorclotting properties of natural fibrin requires that these requisitephysiologic conditions be met. The FS of the present invention is in oneembodiment is activated by the mixing of two activated orself-activating components at the time of delivery. The first componentis a concentrated fibrinogen preparation, which in certain embodimentsfurther comprises a protease inhibitor, such as aprotinin. This is mixedequally with the second activator component comprising thrombin or athrombin-like equivalent and calcium, such as CaCl₂, although the secondactivator components maybe sufficiently available at the wold site thatadditional components are not needed. Each of the two components, andany compound added thereto, is selected and prepared to ensure thatphysiologic fibrinogenesis is duplicated as closely as possible in theFS product.

Nonlimiting examples of fibrin monomers include fibrin I monomer, fibrinII monomer or des BB fibrin monomer, or combinations thereof.Technically the term “FS composition” is used to refer to the mixture ofthe fibrinogen and activated thrombin or thrombin-like activator (aswell as other essential and/or additive components) in the secondsbefore a fibrin clot forms. Once the FS fibrin clot has irreversiblyformed, the term “FS product,” or simply “fibrin sealant” or “FS” areherein used. Nevertheless since the transition from FS composition to FSproduct occurs within only a few seconds, and since it is a continuousclot forming process, without a bright line change from “composition” to“product,” the terms are essentially interchangeable. More importantly,the terms are used to indicate the temporal transition from mixture ofcomponents in the FS composition to final FS clot in the resulting FSproduct.

Also, for the purpose of the subject invention, “fibrin polymer”includes any polymer resulting from the polymerization of fibrinmonomer. Thus, for example, the conversion of fibrin I monomer to fibrinpolymer can result in fibrin I polymer, cross-linked or noncrosslinked,and/or fibrin II polymer, cross-linked or non-cross-linked, depending onhow the conversion step is carried out.

The viscosity of the components of the FS composition, as well as thatof the FS fibrin preparation formed upon activation of the FScomponents, can be varied so that delivery, positioning, and stabilityduring polymerization are appropriate to provide the necessary sealingcapability, elasticity and strength for the selected FS application.Such attributes allow faster, more efficient surgical repair of damagedor weakened tissues than is possible with suturing or known suturelessprocedures. The FS product, preferably delivered to the wound site inthe form of a solution, most preferably as an aqueous solution, mustprovide the tensile strength necessary to keep the welded tissuetogether, joining the separated tissue or providing a watertight,flexible seal on a tissue, or prosthetic or implant surface.

Optionally, a viscosity modifier and/or bonding enhancer may be added asdescribed below to the composition according to need. The resultingcomposition provides a FS product having excellent strength and superiorhandling characteristics. The composition is particularly suited forlaser welding by forming a strong, uniform, elastic weld or coating.

The FS composition of the present invention comprises protein componentsselected from natural or synthetic peptides, including full-lengthmolecules, enzymatically active modified, cleaved, or shortened variantsthereof, or cross-linked derivatives thereof (Coller et al., J. Clin.Invest. 89:546-555 (1992)), as well as mixtures thereof. Included amongthe peptides are simple proteins, conjugated proteins, and mixturesthereof. Examples of such proteins include globular proteins and fibrousor structural proteins. Examples of globular proteins include syntheticor natural serum proteins, natural or synthetic derivatives thereof,salts, enzymatically, chemically, or otherwise modified, cleaved,shortened or cross-linked, oxidized or hydrolyzed derivatives orsubunits thereof, and mixtures thereof.

The FS composition is prepared in a form ranging from a flowable liquidto a sol to a viscous gel depending upon the application and theconcentration of components. For example, the composition is preferablyemployed in the form of a viscous gel for bonding separated tissues,wherein the gel quickly polymerizes into a durable, water insoluble,irreversibly cross-linked clot to secure the tissues together. On theother hand, the formation of a. watertight or resistant seal on tissuesor prosthetic materials may be most efficiently accomplished using aless viscous composition. In some cases activation of the storage-stablefibrinogen component will spontaneously form a weld. In other cases, itmay be necessary to activate the composition, with energy and/orphotons.

Components of Instant Fibrin Sealant

Storage-Stable Fibrinogen Component

The characteristics if the FS composition of the present invention aredefined and distinguished from prior art compositions that may be usedfor similar applications by the nature of the components from which thesubject FS is prepared. The primary component of the preferredembodiment of the present FS invention is a highly concentrated solutionof fibrinogen. Key to the present FS composition is its shelf-readyinstant availability for its intended purposes, which is enabled by itsformulation using a storage-stable, ready-to-use aqueous fibrinogencomponent, see e.g., U.S. patent application Ser. Nos. 10/267,104 and10/263,987, the contents of which are herein incorporated by reference.

The storage-stable fibrinogen component may be originally prepared fromany fibrinogen preparation, whether isolated and purified from bloodplasma, produced by cell-culture techniques, recombinantly prepared, orfreshly isolated, or freshly prepared from a lyophilized or deep-frozenplasma-derived preparation. Regardless of the source, the fibrinogenpreparations are handled and used in essentially the same way onceconcentration and components remade equivalent. The storage stability ofthe fibrinogen component is irrelevant to the original source of thefibrinogen; in fact, it is the storage method and conditions of theaqueous solution that cause the fibrinogen solution to remain stable,while others have failed to create a suitable storage-stable fibrinogensolution.

After addition of thrombin/Ca⁺⁺ to the ready-to-use fibrinogen solution,the rapid increase in viscosity and decrease in liquid movement that isseen, is referred to as a “gel.” In the gel state, the fibrinogensolution no longer flows freely, but can be forced to move withagitation. Although this measurement is subjective, the estimatedvariability is only ±2 seconds.

“Clot” formation is the sudden solidification of the fibrinogensolution, beyond which agitation cannot force liquid to flow from thesolidified material. The immobile material usually becomesmacroscopically opaque white and viscoplastic. Scanning electronmacrographs (SEM) photographs of typical physiological ornon-physiological fibrin clots are shown, for example, in Redl et al.,Medizinische Welt 35:769-76 (1985). A clot generally adheres to a testtube wall and cannot be dislodged by sharp tapping of the tube on asolid surface. This measurement is less subjective than gel formation,and estimated uncertainty is only ±1 second for rapidly setting samples(8-12 seconds), although it may be slightly greater for slower clotting(>100 seconds) samples.

Preparing the Fibrinogen Component

When the fibrinogen component is prepared from a lyophilized ordeep-frozen plasma-derived preparation, the length of time thefibrinogen preparation has been lyophilized or deep-frozen is not afactor in the preparation of the FS composition of the presentinvention, so long as the biological activity of the freshly preparedfibrinogen solution is equivalent to a comparable sample of isolated andpurified fibrinogen from fresh plasma, and spontaneous clotting has notbeen induced in the solution.

When the fibrinogen component is prepared from whole blood, typically avolume of blood, such as 100 ml, is collected into a standardcommercially available blood bag containing an anticoagulant. Anyanticoagulant can be used, such as, without limitation, heparin, EDTA,hirudin, citrate or any other agent that can, directly or indirectly,prevent the formation of thrombin. Citrate is preferred, and is commonlyfound in commercially available fibrinogen preparations. The plasma,which contains the fibrinogen component, is then separated from thewhole blood.

Currently available, commercial fibrinogen contains salts used in theisolation and purification process. As noted in the Examples, thisincludes sodium citrate and sodium chloride, but presence of such saltsthat are a residual part of the fibrinogen purification process do notappear to affect the storage-stability of the resulting preparation orits effectiveness in the preparation of the present FS composition.Since the storage-stable, ready-to-use fibrinogen solution is onlyeffective if it retains the characteristics of a comparable, freshlyprepared fibrinogen solution, the effect of the fibrinogen purificationprocess is essentially the same for both and not relevant to the presentinvention. Nevertheless, extremely high concentrations of citrate and/orsodium may affect clotting of the stored fibrinogen preparation.

Nonlimiting sources of FS components are blood, preferably mammalianblood and even more preferably human blood, cell cultures that secretefibrinogen and recombinant fibrinogen, with blood plasma beingpreferred. Blood can be any form of blood including, for example, wholeblood. Also, blood can be utilized to prepare an autologous fibrinsealant (from the patient's own blood products). Autologous fibrinogencan be prepared and stored for later use by the human or veterinarypatient using the storage-stable methods of U.S. patent application Ser.Nos. 10/267,104 and 10/263,987.

Any separation technique can be utilized, for example, sedimentation,centrifugation or filtration. For example, using centrifugation, theblood is transferred to a container suitable for centrifugation andcentrifuged at room temperature for 10 minutes at 3,000 g. The clearsupernatant plasma (approximately 50 ml) is decanted and the cellularcomponents are discarded. However, if it is desired to obtain plasmarich in platelets, centrifugation can be carried out at lower g force,e.g., 500 g for about 20 minutes. The supernatant, which contains theplasma, can be removed by standard techniques. Fibrinogen is thenisolated from the resultant plasma and treated to preserve stability,e.g., in accordance with U.S. patent application Ser. Nos. 10/267,104and 10/263,987, until needed to prepare the FS of the present invention.

In one embodiment, the plasma-derived fibrinogen component is preparedfrom whole blood by filtration. Filtration can be carried out by passingthe whole blood through a suitable filter that separates blood cellsfrom plasma. It is preferred that the filter be a microporous membraneexhibiting good protein transmission. As above, 100 ml whole blood iscollected into a bag containing a suitable anticoagulant, then the bloodis then recirculated over a filter exhibiting good protein transmissionby means of peristaltic pump. The pressure drop across the membraneresults in plasma being forced through, while cellular components remainin the recirculating blood. Plasma (50 ml) is collected for furtherprocessing as described above.

In an alternative embodiment, any cell culture that can secretefibrinogen can be utilized in the subject invention. The culture andmaintenance process is carried out essentially as described by standardtexts on mammalian cell culture. For example, HEPG2 cells may be usedfor this purpose (see, e.g., Liu et al., Cytotechmology 5:129-139(1991)). The cells are seeded into flasks at a split ratio between 1:4to 1:8 in Minimal Essential Medium containing 10% calf serum andbuffered with 5% CO₂ and maintained at about 37° C. After 24-36 hoursthe medium is removed and replaced with serum free medium containing asuitable protease inhibitor and 2 IU/ml heparin. Culture is continuedfor additional 24 hour periods, with three consecutive changes of serumfree media. The conditioned media is centrifuged at 3,000 g for 10minutes to remove any cell debris and the clarified supernatant containsfibrinogen, which can be further concentrated as desired using knownmethods.

The fibrinogen component of the present FS composition can also beprepared from recombinant DNA techniques (see, e.g.; Roy et al, J. Biol.Chem. 266:4758-4763 (1991)). Roy et al. teach methods for expressing allthree chains of fibrinogen and teach that COS cells express, assembleand secrete the chains in a form that is capable of forming athrombin-induced clot. Once prepared, the cellular debris is removed bycentrifugation or filtration as described above as used for cellcultures, and then the fibrinogen may be concentrated.

The preparation by cell culture or recombinant techniques of thestorage-stable fibrinogen component used in the present invention may bepreferred in certain embodiments because viral contamination by plasmacontaminants is eliminated, and there is more complete control over thepresence of other components in the final FS. For example, Factor XIIIis often present in fibrinogen preparations from plasma. However, unlessexpressly added, no Factor XIII is present in fibrinogen prepared bycell culture, permitting the amount added to the FS composition to causecross-linking of the fibrin strands to be precisely quantified.

The preferred embodiments of the invention are applicable to a crudefibrinogen product in the course of preparation, or to a final,concentrated fibrinogen preparation having greater than 90% proteinpurity and being greater than 95% clottable protein, or to anyconcentration of fibrinogen there-between. For instance, in the Examplesthat follow, the human fibrinogen preparation had 53% protein purity and95% clottable protein, while the bovine fibrinogen preparation had 61%protein purity and 97% clottable protein. Nevertheless, both wereapplicable to preparation of the FS composition of the presentinvention.

In a preferred embodiment of the invention, the storage-stablefibrinogen preparations of the present invention, although highlyconcentrated, remain solubilized in aqueous solution making thefibrinogen particularly suitable for use in the preparation ofsupplemented or unsupplemented, ready-to-use FS compositions. Thefibrinogen is optimally stored at a concentration of 10-85 mg/ml, morepreferably at a concentration of 15-75 mg/ml, even more preferably at aconcentration of 30-70 mg/ml, and most preferably at a concentration of40-65 mg/ml when is used to prepare a ready-to-use FS composition.Moreover, the concentration of fibrinogen, or fibrinogen-containingprotein, in the storage-stable aqueous solution of the present inventiongenerally ranges from 2 to 10 w/v %. preferably 4-7 w/v %. Theconcentration of fibrinogen is determined by protein absorbencemeasurements at 280 nm (using 14 as the extinction of 1% fibrinogensolution).

In the preferred embodiments of the invention, storage-stable fibrinogenis biologically active (i.e., clot in the presence of thrombin and Caions), and have essentially the same physical characteristics as freshsamples. This produces the same type of controlled fibrin clot formedusing freshly prepared fibrinogen when FS compositions are prepared andused. Fibrinogen (and thrombin) concentrations dictate time to clotformation, clot strength, clot adhesion, and thus hemostasis. For thepurposes of discussion, this type of clot is referred to herein simplyas a “fibrin clot” to differentiate the process from a “spontaneousclot,” wherein the latter may occur in an unstable, concentratedfibrinogen solution, even absent thrombin or another activator.

However, the terms are used herein only for the purpose ofdistinguishing the FS compositions prepared from the storage-stablefibrinogen solutions in which the activity of the composition is quicklydemonstrated byte rapid formation of a fibrin clot when equal amounts ofthe fibrinogen and thrombin/Ca⁺⁺ are vigorously mixed, from aspontaneous clot which is indicative of instability in the prior artfibrinogen solutions. The fact that prior art, aqueous solutions offibrinogen are known to be highly unstable, and tend to spontaneouslyclot upon storage, makes the storage of fibrinogen in ready-to-useliquid form impractical for even a day or two using previouslyrecognized methods.

In preferred embodiments of the invention, prior to its use, thestorage-stable fibrinogen is stored in a polymer, plastic orplastic-based container, although more preferably the plastic containeris polypropylene. Glass is not to be used to store fibrinogen orplatelets because glass enhances spontaneous clot formation.

The fibrinogen solutions of the present invention are ideally suited forforming a physiological fibrin structure when exposed to an activatorsolution, and fibrin clots are rapidly formed. This is proven by mixingthe stored fibrinogen solution with an equal volume of a thrombin/CaCl₂solution (comprising, e.g., 2.5 units/mg fibrinogen (100 units/ml)thrombin and 3-6 mM excess CaCl₂ over citrate or other chelators thatmay be added to solutions), as set forth below. If the resulting clotdemonstrates a physiological fibrin structure, it will have the typical,spatial branched fibril structure shown when clots are formed by theaction of thrombin on freshly-prepared or freshly isolated and purifiedhuman fibrinogen under physiological conditions, i.e., at an ionicstrength of approximately 0.15 and approximately neutral pH.

Prior experiments have proven, by continuous observation and testing,that the aqueous fibrinogen solutions of the invention under thepreferred conditions remain stable (active and not spontaneouslyclotted) for at least 97 days at pH 6.3 to 8.0, when stored at roomtemperature (˜23° C.) or refrigerated (˜4° C.). In fact, the componenthas been shown to be stable for extremely long periods of time, ascompared with known deep frozen or lyophilized preparations of theconcentrated protein that have been maintained without a substantialloss of activity (i.e., fibrinogen/thrombin fibrin clots are stillrapidly formed upon mixing), even years after the initial storage of thefibrinogen product. Thus, “long term storage” means storage of thefibrinogen solution, preferably human fibrinogen solution, inready-to-use form under the presently disclosed conditions, withoutsubstantial loss of protein activity for at least 3 days, preferably forat least 3 weeks, more preferably for at least 10 weeks, more preferablystill for at least 6 months, even more preferably for at least 1 year,and yet more preferably for at least 2 years (assuming it is frozen for≧1 year, and then stored at ˜4° for ≧an additional year). Therefore,under optimal conditions the fibrinogen solution will remain stable forperiods at least or greater than 2 years.

Although it is preferred to use “human” fibrinogen in FS applications inaccordance with the methods of the present invention for a humanpatient, the use of stabily stored, ready-to-use, aqueous fibrinogensolutions from other species, most preferably species of other mammals,is also applicable. In fact, there appear to be no species compatibilityissues associated with the use of the stored human fibrinogen with amammalian species. For example, the subject human fibrinogen maybe usedfollowing storage in aqueous solution to prepare, e.g., a biologicallycompatible tissue adhesive preparation for use in or on any species ofmammal. However, it is understood that an advantageous application ofthe present human fibrinogen preparation results from its ready-to-useapplicability to human subjects.

Fibrinogen Storage Conditions: Temperature and pH

The optimal temperature and pH of the storage-stable fibrinogencomponent would be known in accordance with the present invention, orboth could be rapidly determined, by one of ordinary skill in the artusing known means. However, aqueous-based gels could also be used in thepresent invention, so long as such material permits the completesolubilization of the fibrinogen contained therein, and so long as thepreparation is sufficiently fluid as to permit the rapid preparation ofready-to-use biological tissue adhesives or other applications followingstorage in accordance with the methods disclosed herein. A key to thepresent FS invention is the fact that the fibrinogen component is storedin ready-to-use fluid form. In its ready-to-use form, it is storedneither as a lyophilized preparation, nor is it in a deep frozen state.

The temperature of the solution during storage is not particularlyrestricted, so long as the fibrinogen contained therein remains stable(i.e., neither inactivated nor spontaneously clotted). The preferredtemperature for storage of the fibrinogen solutions of the presentinvention ranges from 1° to 25° C., more preferably from about 4° toabout 23° C. When refrigerated, the optimal temperature is about 4°C.±1° C., at which temperature the product has proven to be stable forat least 1 year (data not shown). When storage is at room temperature,the optimal temperature ranges from about 20° to 25° C., more preferablyfrom about 22° to 24° C., most preferably the temperature is about23°±1° C., at which temperature the product has proven to be stable forat least 3 months (data not shown). Moreover, previously frozen samples(for up to at least 1 year) have been subsequently stably stored at 4°C. for at least an additional year, making the product available for useherein in ready-to-use form for at least 2 years.

The pH value of the aqueous fibrinogen solution is preferably adjustedduring storage to approximately pH 6.0 to 8.2, more preferably pH6.2-8.0, even more preferably pH 6.3-7.5, and most preferably pH 6.5 to7.36 and exemplified at pH 7.24 for bovine fibrinogen, and mostpreferably pH 6.32 to 7.13 for human fibrinogen.

The pH of the storage-stable fibrinogen solution is determined by thebuffer in which it is stored. In a preferred embodiment of the inventionstorage-stable fibrinogen solutions are prepared in histidine buffer,although other recognized, physiologically acceptable buffers known tothe art may be used to prepare the storage-stable fibrinogen, so long asthe resulting pH of the fibrinogen solution remains within theproscribed range, such that its activity is maintained, but it remainsfree of spontaneous clotting. Suitable buffers, e.g., 0.1 M, include butare not limited to achieve the pH levels such as those that are noted:histidine, pH 6.0 or 7.2 to 7.24; Tris pH 8.16; glycine pH 9.3; orcarbonate, pH 9.05 to 9.31 or pH 9.86 to 9.9.

The optimal pH for the storage of a particular fibrinogen solutiondepends in part upon the temperature at which the material is to bestored, as is shown in the Tables that accompany the Examples whichfollow. However, in light of the information provided herein, one ofordinary skill in the art would be able to select the optimal pH for thefibrinogen solution based upon the planned storage temperature andconditions, knowing that the determining factor is whether the proteincontained therein remains stable (i.e., neither inactivated norspontaneously clotted).

For example, ready-to-use human fibrinogen stored at room temperature(˜23° C.) is optimally maintained at pH 6.3 to 7.1, preferably atapproximately pH 6.32 to retain the ability to rapidly form a clot whenthe stored preparation is neutralized and exposed to thrombin/Ca⁺⁺. Whenready-to-use human fibrinogen is stored under refrigeration (˜4° C.) theoptimal pH is also optimally maintained at pH 6.32 to 8.0, preferably atapproximately pH 6.3 to 7.5 to retain the ability to rapidly form the FSclot when the stored preparation is neutralized and exposed tothrombin/Ca++(see Table 2).

Similarly, ready-to-use bovine fibrinogen stored at room temperature(˜23° C.) is optimally maintained at pH 6.5 to 9.0, preferably atapproximately pH 6.5 to 8.2, to retain the ability to rapidly form aclot when the stored preparation is neutralized and exposed tothrombin/Ca⁺⁺. When ready-to-use bovine fibrinogen is stored underrefrigeration (˜4° C.), the optimal pH is also optimally maintained atpH 6.5 to 9.0, preferably at approximately pH 6.5 to 8.2, morepreferably at pH 6.5 to 7.07 to retain the ability to rapidly form aclot when the stored preparation is neutralized and exposed tothrombin/Ca⁺⁺ (see Tables 1 and 2).

Activator Component, e.g., Thrombin or Thrombin-Like Enzyme

The “activator” of the present invention is thrombin or a thrombin-likeenzyme. A “Thrombin-like enzyme,” including thrombin, means any enzymethat can catalyze the formation of fibrin from fibrinogen. In additionto thrombin from mammalian, blood sources, preferably from human sourcesfor use with human patients, the enzyme can be produced by cell cultureor recombinant means and isolated as described below regarding thefibrinogen component. Bovine thrombin is conveniently and commerciallyavailable from a variety of sources, including Parke-Davis.

Thrombin acts as a catalyst for fibrinogen to provide fibrin, aninsoluble polymer. Thrombin is present in the FS composition in anamount sufficient to catalyze polymerization of fibrinogen. Thrombinalso activates Factor XIII, a plasma protein that catalyzes covalentcross-links in fibrin, rendering the resultant clot insoluble.

As an alternative to thrombin or thrombin analogs, a common source ofthrombinlike enzymes are purified from the reptilase coagulants i.e.,snake venoms (see, e.g., Pirkle et al, Thrombosis and Haemostasis,65(4):444-450 (1991)). A preferred thrombin-like enzyme is, withoutintended limitation, ancrod or batroxobin, especially from B. Moojeni;B. Maranhao and B. atrox and Ancrod, especially from A. rhodostoma.Depending on the choice of thrombin-like enzyme, such thrombin-likeenzyme can release fibrinopeptide A, which forms fibrin, although atdifferent rates than thrombin.

It should be noted that if the storage-stable fibrinogen component ofthe present FS preparation comes into contact with the patient's blood,e.g., at the wound site, the patient's own thrombin and Factor XIII maybe sufficient to convert the fibrin polymer to cross-linked fibrinpolymer. Thus, endogenous prothrombin and Factor XIII can be utilized inthe FS of the subject invention as components of the compositioncomprising fibrin monomer or non-cross-linked fibrin. However, it shouldbe noted that sufficient quantities of endogenous thrombin and FactorXIII are typically not retained in amounts sufficient to convert thestorage-stable fibrinogen component to cross-linked fibrin at a reactionrate that is suitable for producing an effective fibrin seal. In largerwounds, the heavier blood flow will wash away the endogenous material,and clotting will not take place. It appears that more thrombin isrequired to convert fibrinogen to cross-linked fibrin than to convertnon-cross-linked fibrin to cross-linked fibrin at an equivalent reactionrate.

The concentration of the thrombin component in the FS composition of thepresent invention can range from as little as 150 μg thrombin per 40 mgfibrinogen in solution to an equal ratio of thrombin and fibrinogen insolution, depending on the application, surrounding conditions (e.g.,temperature, pH, mixing), and the rate of polymerization desired. Interms of the FS composition rather than the fibrinogen component, fromabout 4 units to about 500 units of thrombin per ml of FS composition isadded. Alternately, the thrombin component can be provided by the woundsite. However, polymerization of the fibrinogen component will proceedmore quickly as more thrombin is available to activate each molecule offibrinogen in solution, up to a maximum at which point increasedpolymerization is not possible by the addition of thrombin alone.

A source of calcium ions, e.g., as CaCl₂, is essential to activate thethrombin component before the thrombin component can activate thefibrinogen component to for the present FS composition. However, thecalcium ions may be incorporated into the stored thrombin component.Alternatively, CaCl₂ may be added to the FS composition prior topolymerization, or sufficient quantities of calcium ions may simply beendogenously available at the wound site.

In Factor XIII-free FS preparations, Factor XIII is optimally added toactivate crosslinking of the fibrin. Activated Factor XIII can be addedto the FS composition at a final concentration of from about 1.0 toabout 20 units Factor XIII per ml of FS composition. Alternatively, theFactor XIII can be supplied by the blood or body fluids at the woundsite, or by the addition of autologous plasma.

In one embodiment of the present invention, the activator enzyme isimmobilized onto a support. This can be carried out by any suitabletechnique. For example, various activation chemistries available forderivatizing supports are: diazonium groups, isocyanate groups, acidchloride groups, acid anhydride groups, sulphonyl chloride groups,dinitro fluorophenyl groups, isothiocyanate groups, hydroxyl groups,amino groups, n-hydroxysuccinimide groups, triazine groups, hydrazinogroups, carbodiimide groups, silane groups and cyanogen bromide. Seee.g., Dean, in Affinity Chromatography—A practical Approach, Johnson andMiddle (Eds) (1991) IRL Press Oxford, the procedures of which areincorporated by reference. Low pH values, e.g., pH 4-6, can be utilizedfor enzyme coupling to prevent enzyme degradation.

Agarose may be used as the support, although it is also possible to usesilica. Generally, The support is activated by a highly reactivecompound, which subsequently reacts with a functional group of a ligand,e.g., —OH, —NH₂, —SH, —COOH, —CHO, to form a covalent linkage.

In certain embodiments of the invention, the FS composition is activatedthrough the application of energy and/or photons. The energy preferablyhas a wavelength in the electromagnetic spectrum, and is selected fromX-rays, ultraviolet light, visible light, infrared light, andradiowaves. Thermal energy delivered through direct contact as, forexample, with a probe heated electrically, such as an electrocautery, ora probe heated through gas compression in the tip, or the passage ofheated gas or liquid through the tip, may be used. Sound energy in theultrasonic frequency, or radiowaves in the microwave range may also beemployed. The energy is delivered in a continuous or noncontinuousfashion, in a narrow or broad band of electromagnetic wavelengths.Examples of photon sources include monochromatic and polychmromaticlight, coherent or noncoherent light, delivered in a continuous ornoncontinuous fashion. Examples of noncontinuons energy and/or photondelivery include single and/or multiple pulse train delivery. Photonscan be delivered in a polarized or nonpolarized fashion, direct orreflected, with or without internal or external interference. In apreferred embodiment, lasers are used, including, but not limited to,those in the ultraviolet, visible, or infrared range.

Stored solutions of ready-to-use human fibrinogen that do not clot whenthrombin and calcium ions are added with vigorous agitation are called“thrombin-insensitive.” The thrombin insensitive preparations remainfluid (having viscosities similar to water). However, analysis of suchthrombin insensitive fibrinogen samples by SDS-PAGE (sodium dodecylsulfate polyacryamide gel electrophoresis) has shown that the fibrinogenprotein has been irreversibly degraded to small molecular weightfragments. Thus, the preparation no longer contains active fibrinogen,and is not the subject of the present invention.

Supplements and Additives

As noted, the FS composition of the present invention can additionallycontain viscosity modifiers and/or bonding enhancers in accordance withthe end use of the composition. For example, the addition of viscositymodifiers provides a FS composition with a viscosity particularly suitedto the tissues being repaired or sealed. A composition having a highviscosity is preferably employed to bond separated tissues while lowerviscosity compositions are best suited to form a coating for watertightsealing of continuous tissue masses and prosthetic materials, such asGortex™ vascular grafts and the like. Such viscosity modifiers include,without limitation, non-cellular matrix materials, such as hyaluronicacid and salts thereof (e.g., sodium hyaluronate or sodium chondroitin);or saccharides, such as fructose, hydroxypropyl-methylcellulose,hydroxyethylcellulose, carboxymethylcellulose, hydroxymethylcellulose,dextrans, agarose, alginic acid or pectins; or polyalcohols, such asglycerin; or protein gels, such as collagen and caseinates; or mixturesthereof.

Bonding enhancers may also be used to improve the bonding strength ofthe composition. Such bond enhancers may be (i) added to the activatorcomponent before mixing with the storage-stable fibrinogen component or(ii) added to the activated fibrinogen/fibrin mixture prior topolymerization, or (iii) spread over the wound surface prior toapplication of the FS material. The bond enhancers are generallyselected from polar compounds, such as charged glycosaminoglycans,oligosaccharides and polysaccharides, polyalcohols, and polar dyes.Notably many of these compounds also operate as viscosity modifiers.Examples of such polar compounds include without limitation, hyaluronicacid, chondroitin sulfate, carboxymethyl-cellulose,hydroxymethylcellulose, glycerin, indocyanine green, and fluoresceinsodium. Polyvalent cations, such as calcium, may also enhance bonding bybinding to the negatively charged moieties in the protein components ofthe FS composition, such as albumin, and glycosaminoglycans, such ashyaluronic acid and chondroitin sulfate.

Mucoadhesives are particularly useflul bond enhancers when the woundsurface contains mucin, such as the gastrointestinal tract and thepulmonary system. Examples of mucoadhesives includecarboxymethylcellulose and sodium alginate. Use of these materials onwound surfaces having a high collagen content, which have a largeconcentration of hydroxyl groups, may also be advantageous infacilitating bond formation. As reported by Robinson et al., Ann. NYAcad. Sci. 507:307-314 (1987)), a high charge density is preferred forboth swelling and hydrogen bonding, thus permitting firm attachment tothe desired tissue surface. Other mucoadhesives as would be obvious toone skilled in the art may also be employed.

The composition may as necessary additionally contain pH modifiers,surfactants, antioxidants, osmotic agents, and preservatives. Examplesof pH modifiers include, for example, without limitation, acetic acid,boric acid, hydrochloric acid, sodium acetate, sodium bisulfate, sodiumborate, sodium carbonate, sodium citrate, sodium hydroxide, sodiumnitrate, sodium phosphate, sodium sulfite, and sulfuric acid. Examplesof surfactants include, for example, benzalkonium chloride. Examples ofantioxidants include, for example, bisulfates. Examples of osmoticagents include, for example, sodium chloride. Examples of preservativesinclude, for example, chlorobutanol, sorbate, benzalkonium chloride,thimerosal, methylparaben, propylparaben, EDTA(ethylenediaminetetraaetic acid), and polyquad.

Typically, pH modifiers, surfactants, antioxidants, osmotic agents, andpreservatives are present in a concentration of from about 0.001 to 5%by weight.

The components of the composition are combined together in quantities,which provide a desired bonding strength, as well as a viscosity, whichis particularly adapted to the intended end use. In general, the amountof the peptide in the FS composition is in the range of from about 1 to99% by weight preferably about 5 to 80% by weight, more preferably about6 to 70% by weight, more preferably still about 6 to 50% by weight toabout 8 to 35% by weight. Saccharides, if present, may be combined inthe range of from about 0.1 to 70% by weight. Glycosaminoglycans, ifpresent, is preferably from about 0.1 to 20% by weight. Polyalcohols, ifpresent, may be added in an amount of from about 0.1 to 90% by weight.

The amount of additives, such as viscosity modifiers and bondingenhancers is generally no more than about 65% by weight.

The viscosity of the FS composition is chosen in accordance with theparticular surgical procedure being performed. For bonding of separatedtissues, a viscosity of from about 1,000 to 1,000,000 centipoise isadvantageous, preferably in the range of from about 20,000 to 200,000centipoise. A FS composition having a viscosity in the preferred rangecan be easily placed on the separated tissues by ejecting through ahypodermic syringe or dual-syringe device, and spreading over the woundarea by moving the syringe tip. Within that viscosity range, the FScomposition does not run off the tissues and remains fixed, even whenenergy is applied to form the tissue weld.

The viscosity of the FS composition or the present invention ispreferably lower for applications requiring the formation of awatertight coating for sealing tissues or prosthetic materials. For suchpurposes, the preferred viscosity for coating is in the range of from 10to 1,000 centipoise. The lower viscosity is preferred to permit theready capability to spread the composition to efficiently cover thetissue or material being coated.

When hyaluronic acid, or other non-Newtonian fluids are added to the FScomposition, the viscosity decreases with increasing shear forces.Accordingly, the viscosity of the FS composition can be modulated byaltering the shear forces when the composition is applied to the woundsurface. For example, a very thick FS composition can be injectedthrough a graft at a rapid or high sheer rate to reduce viscosity duringthe transit phase in which the graft is coated with the materialapplying a property known as pseudoplasticity, This makes the highlyviscous FS material ideal for welding at sites that are not subject toshearing forces during the polymerization process. When the compositionis injected, shear forces are high, and viscosity decreases, permittingeasy injection. After being deposited on the tissue, the shear forcesdrop to zero, and the viscosity of the composition rapidly increasesaccordingly. As a result, the composition remains localized on thetissue.

In certain embodiments of the present invention, the FS composition issupplemented with, and acts as a carrier vehicle or delivery vehiclefor, any number of compounds, alone or in combinations of two or more,for example, but not limited to, growth factors, drugs, blood factors orother compound(s) or mixtures thereof, so long as noted above, theactivity of the fibrinogen solution is maintained throughout the lengthof the storage and spontaneous clotting is not induced. For instance, bysupplementing the FS composition, or one component of the FScomposition, such as the storage-stable fibrinogen component with agrowth factor, the FS composition can, when applied to a human patientor animal subject particularly at a wound site, accelerate, promote orimprove wound healing, tissue (re)generation, and the like.

The supplement may be mixed with the fibrinogen or thrombin component,or with a combination thereof, or with the final FS composition,depending on the nature of the additive, the FS polymerization rate,interaction between components, and the like. It is believed that thedosage of such supplements is the same as that utilized in conventionalfibrin sealants.

Optimally, the supplemented FS composition when used as a carrier ordelivery vehicle acts to: (1) potentiate, stimulate or mediate thebiological activity of the growth factor(s), ding(s), or otheradditive(s) or component(s); (2) decrease the activities of one or moreadditive(s) or component(s) of the supplemented FS composition orstorage-stable fibrinogen component used therein, wherein suchactivities would otherwise inhibit or destroy the growth factor(s) inthe preparation; (3) allow prolonged delivery of the additive orcomponent from the FS composition or the storage-stable fibrinogencomponent of the present invention; and (4) possess other desirableproperties. The contemplated additive(s) or supplement(s) are intendedto also include any mutants, derivatives, truncated or other modifiedforms thereof, which possess similar biological activity(ies), or asubset thereof, to those of the compound or composition from which it isderived.

More than one additive or component may be simultaneously added to orsupplied by the FS composition of the present invention. Although theconcentration of such additive(s) and/or component(s) will vary in theFS composition depending on the objective, the concentration must besufficient to allow such compound(s) and/or composition(s) to accomplishtheir intended or stated purpose. The amount of such supplement(s) to beadded can be empirically determined by one of ordinary skill in the artby testing various concentrations and selecting that which is effectivefor the intended purpose and site of application. Dyes, tracers, markersand the like may also be added, for example, to examine the subsequentdelivery of the FS composition.

Supplemented FS preparation may comprise, e.g., drug(s), antibody(ies),anticoagulant(s), coagulation factors such as Factors VII, VIII, IX, Xand XIII, and von Willebrand's factor, as well as growth factors, and/orother compounds that are presently delivered to a human or animalpatient in need of such by other delivery devices or mechanisms that maynot operate as efficiently or effectively as the present invention.

Various components may be added which serve to recruit or expand theleukocyte or endothelial population, inhibit pathways of leukocytes,endothelial cells or the like, or to modulate novel peptides. Compoundsof biological value include, without limitation, growth factors, e.g.EGF, TGFα, TGFβ, TGF-I and TGF-II, FGF, PDGF, etc.; cytokines, e.g.,IFN-α, IFNβ, IL-2, IL-2, IL-3, IL-6, hematopoietic factor, etc.;immunoglobulins; metabolic substances, e.g., insulin, corticosteriods,hormones, etc. Other materials include structural materials, such asphysiologically acceptable alloplastic materials, e.g., polymers,glasses, metals, ceramics, composites thereof, etc.

Other materials can also be added, for example, fibronectin,fibrinolytic inhibitors, such as aprotinin, alpha-2 antiplasmin, PAI-1,PAI-2,6-aminohexanoic acid, 4-aminomethyl cyclohexanoic acid, orcollagen.

The FS material may be mixed with cells, autologous, cultured ormodified, allogeneic or xenogeneic, such as epithelial, epidermal,fibroblast, osteoblast, mesenchymal, hepatic (hepatocytes), pancreatic(e.g., macrophage, platelet, T-cell, B-cell, granulocytes, monocytes,keratinocytes, etc.), or cultured modified cells, to deliver therapeuticor growth enhancing substances.

For dental or orthopedic applications, inorganic minerals or a mixtureof inorganic minerals, naturally occurring or synthetic, desirablyhydroxyapatite or minerals found in bone powder or chips may be added tothe formulation. The mineral(s) are present in a volume ratio to thefibrinogen component of from about 1:2 to about 4:1 depending upon thedesired flow characteristics or intended use and site. Demineralizedbone matrix (DBM) is a source of osteoinductive proteins, known as bonemorphogenetic proteins (BMP), including osteogenin, and growth factorswhich modulate the proliferation of progenitor bone cells (see, e.g.,Hauschka et al., J. Biol. Chem. 261:12665-12674 (1986) and Canalis etal., J. Clin. Invest. 81:277-281 (1988)). Unfortunately, DBM materialshave little clinical use unless combined with particulate marrowautografts, and there is a limit to the quantity of DBM that can besurgically placed into a recipient's bone to produce a therapeuticeffect. In addition, DBM powder and osteogenin may be washed away bytissue fluids before their osteoinductive potential is expressed.Moreover, seepage of tissue fluids into DBM-packed bone cavities orsoft-tissue collapse into the wound bed are two factors that maysignificantly affect the osteoinductive properties of DBM andosteogenin. Soft-tissue collapse into the wound bed may likewise inhibitthe proper migration of osteocompetent stem cells into the wound bed.

FS also can serve as a “scaffold,” which cells can use to move into awounded area to generate new tissues. Additionally, viable osteoblastsmay be harvested from a donor site and incorporated into the compositionfor use in transplantation. Other bone restorative materials inparticulate form may be used. Among the suitable alloplastic materialsare polylactic and polyglycolic acids, polymethycrylate, polyHEMA,bioglass, cerevital and other glasses, Al, Ti, CoCr and other metals,Al₂O₃ and other ceramics, etc., and combinations and composites thereof.They may be used in the same volume to volume ratios as for bonemineral. Other restorative materials, such as proteinaceous particles orbeads made from collagen, fibrin, fibrinogen, albumin, etc., may be usedas well, depending upon the tissue repair site. Liposomes may also beincorporated.

As previously noted the FS composition may additionally contain anantibiotic to reduce or prevent infection, e.g., gentamycin, cefotaxim,nebacetin and sisomicin, histaminine H₂-antagonists, e.g., ranitidine,and anticancer drugs (see, e.g., Gersdorff et al., Laryngoscope95:1278-80 (1985); Ederle et al., Ital. J. Gastroenterol. 23:354-56(1991); Ronfard et al., Burns 17:181-84(1991); Sakurai et al., J.Control. Release 18: 39-43 (1992); Monden et al., Cancer, 69:636-42(1992); Greco, J. Biomed. Materials Res. 25:39-51 (1991.); Kram et al.,J. Surgical Res. 50:175-178 (1991)). The antibiotic may be incorporatedinto a liquid component of the FS or into the resulting FS compositionprior to polymerization, if the antibiotic is a liquid, or suspended inthe liquid component, if it is in powder form. The therapeutic doselevels of a wide variety of antibiotics for use in drug release systemsare well known (see e.g., Biomaterials, G. D. Winter, D. F. Gibbons, H.Plank (Eds.), John Wiley & Sons, New York (1980), pp. 669-676).Anti-microbial agents are particularly useful for compositions appliedto exposed wound repair sites, such as sites in the mouth, or tocompromised wound sites, such as burns.

Chromophores and Indicator Compositions

In one embodiment, the composition of the present invention furtherincludes endogenous or exogenous chromophores to facilitatevisualization of the material during placement into warm bloodedanimals. Use of a chromophore allows visualization of the FS fortargeting to the wound site. It also provides a rapid means foridentifying any material that is displaced from the desired applicationsite, and permits subsequent removal of the extraneous material using acellulose sponge, gauze pad, or other absorbing material. The use ofendogenous chromophores, such as hemoglobin, is disclosed in Krueger etal, Lasers Surg. Med. 5:55-60 (1985)). The use of exogenous chromophoresto aid in the placement of biological adhesives has been previouslydescribed (see, e.g., Nasaduke et al., Ann. Ophth. 18:324-327 (1986)).

Chromophores that may be used, include, but are not limited tofluorescein isothiocyanate, indocyanine green, silver compounds such assilver nitrate, rose bengal, nile blue and Evans Blue, Q-Switch™ (Kodak,Inc.), Sudan III, Sudan Black B and India Ink. The chromophores arepreferably present in a concentration of from about 0.01 to 50% byweight based on the total weight of the composition. Other chromophoresof types obvious to one skilled in the art may also be employed.

Such substances may also alter absorption characteristics of thecomposition so that the composition absorbs energy at low energy levels.This enables the heating of the material using certain wavelengths ofthe electromagnetic spectrum which are selectively absorbed by theenergy absorbing compound. For example, this would allow heating of thematerial using certain lasers whose energy would otherwise not beabsorbed by the composition of the present invention, and allows thecomposition to be bonded to the target using these lasers. The selectionof dyes having a peak light absorption at a specific wavelength andmatching tat to the wavelength of light emitted from a light source,such as a laser beam, allows for the selective activation of thecomposition at the site of the coating or seal, while substantiallyreducing the risk of undesirable collateral thermal damage to adjacenttissues.

Exogenous dyes, such as indocyanine green or fluorescein, and endogenouschromophores, such as hemoglobin and melanin, and the like, areparticularly suited for this purpose. These dyes also may increaseadhesivity, bond strength and/or viscosity. Such dyes are preferablypresent in the composition in an amount of from about 0.01 to 50% byweight based on the total weight of the composition.

Chaotropic Agents

In the event that it is desirable to delay formation of the FS productfibrin, a chaotropic agent is added to prevent spontaneouspolymerization of the fibrin monomer, which is formed upon contact ofthe fibrinogen with the activator. The chaotropic agent is mixed withsuch fibrinogen composition and then agitated for about 1 to 2 minutesto form the modified fibrinogen solution. The fibrinogen can then beconverted to a fibrin monomer as described above, but polymerizationwill be delayed.

Suitable chaotropic agents include, for example, urea, sodium bromide,guanidine hydrochloride, KCNS, potassium iodide and potassium bromide.The preferred concentration of the chaotropic agent is from about 0.2 toabout 6.0 molar and most preferably from about 0.3 to about 2.0 molar.It is preferred to utilize the least amount of chaotropic agent possiblethat still prevents the fibrin monomer from spontaneously polymerizing.More preferably a source of calcium ions should not be added to thechaotropic agent until polymerization of the fibrin monomer is desired.This ensures that the fibrin monomer will not cross-link due toactivation by endogenous blood coagulation factors.

If the chaotropic agent was added to the aqueous buffet of thefibrinogen or thrombin components, then the resulting fibrin compositioncan be converted to cross-linked fibrin by diluting the compositionwith, for example, distilled water. The dilution is carried out, suchthat the minimal amount of diluent is utilized. Generally, the resultingconcentration of the chaotropic agent after dilution should be fromabout 0.5 to about 0.1 molar.

Buffering the FS Composition

Upon application to the wound site the FS composition is in oneembodiment preferably buffered to acidity using an acid buffer having apH of less than about 5. Nonlimiting examples of suitable acidic buffersolutions include acetic acid, succinic acid, glucuronic acid, cysteicacid, crotonic acid, itaconic acid, glutamic acid, formic acid, asparticacid, adipic acid and salts thereof. Succinic acid, aspartic acid,adipic acid and salts of acetic acid are preferred, and sodium acetateis more preferred. The preferred concentration of the acid buffer rangesfrom about 0.02 M to about 1 M, more preferably from about 0.1 M toabout 0.3 M. Such preferred concentration renders the ionic strength ofthe composition more biologically compatible.

Accordingly, in one embodiment of the present invention, the compositioncomprising fibrin monomer is substantially free of the activator enzyme.By “substantially free” is meant either that all of the thrombin orthrombin-like enzyme has been removed, or that any thrombin-like enzymeremaining in the composition is at levels insufficient to provide anundesired pharmacological effect. Thus, compositions of this inventionthat are substantially free may contain an activator enzyme in an amountbetween about zero and 10% of the enzyme normally found in a fibrinclot, and preferably between about zero and 2% of the enzyme.

A preferred embodiment of the present invention further provides methodsof preparing the subject instant FS composition in accordance with thepreceding definition.

In yet another preferred embodiment, the FS composition of the inventionis prepared using a suitable alkaline buffer. Nonlimiting examples ofsuitable alkaline buffers include HEPES, sodium hydroxide, potassiumhydroxide, calcium hydroxide, bicarbonate buffers such as sodiumbicarbonate and potassium bicarbonate, tri-metal salts of citric acid,salts of acetic acid and salts of sulfuric acid. Preferred alkalinebuffers include: 0.5.-0.75M sodium carbonate/bicarbonate pH 10-10.5,0.5-0.75M sodium bicarbonate/NaOH pH 10.0, 1.5M glycine/NaOH pH 10.0,0.5-1.0 M bis hydroxeythylaminoethane sulphonic acid (BES) pH 7.5, 1Mhydroxyethylpiperazine propane sulphonic acid (EPPS) pH 8.5, 0.5 Mtricine pH 8.5, 1M morpholino propane sulphonic acid (MOPS) pH 8.0, 1Mtrishydroxymethyl aminoethane sulphonic acid (TES) pH 8.0 and 0.5Mcyclohexylaminoethane sulphonic acid (CHES) pH 10.0; with 0.5-0.75Msodium carbonate/bicarbonate pH 10-10.5, 0.5-1.0M bishydroxeythylaminoethane sulphonic acid (BES) pH 7.5, 1Mhydroxyethylpiperazine propane sulphonic acid (EPPS) pH 8.5 and 1Mtrishydroxymethyl aminoethane sulphonic acid (TES) pH 8.0 being mostpreferred.

The amount of alkaline buffer that is utilized should be enough topolymerize the fibrin. It is preferred that about 10 parts to about onepart of composition comprising fibrin monomer be mixed with about 1 partalkaline buffer. It is even more preferred that such ratio be about 9:1,although the preferred ratio depends on the choice of buffer and thedesired “strength” of the fibrin polymer. Of course, the desiredstrength of the fibrin polymer is determined by the intended end-use ofthe FS composition.

Activating FS Polymerization

In addition to raising the pH or diluting the chaotropic agent of thecomposition comprising fibrin monomer, it is preferred that theprothrombin and Factor XIII of such composition be activated to form thecross-linked fibrin. Such activation can be carried out by thecontacting the components of the FS composition with a source of calciumions. The source of the calcium ions can be included with the fibrinogenor thrombin buffer. As noted above, nonlimiting sources of calcium ionsinclude calcium chloride, preferably at a concentration of about 30 mM.Alternatively, although less preferred, the source of calcium ions canbe supplied by the blood at the wound site.

If a carbonate/bicarbonate buffer is used, the source of calcium ionsmust be added to the acid buffer during the solubilization step. This isbecause calcium chloride is not soluble in a carbonate/bicarbonatebuffer. Preferably, the concentration of calcium ions in the acid buffersolution is from about 5 millimolar to about 150 millimolar, and morepreferably from about 5 mM to about 50 mM.

Product Safety

Unless prepared by cell culture, FS compositions comprise blood plasmaproteins, and as a result are accompanied by a risk of contaminationwith blood-borne pathogens, such as those possibly contaminating humanplasma proteins, in particular, hepatitis viruses or HIV. Using knownviral inactivation methods there have been no reports of viraltransmission from commercial fibrin sealants, even when used on largebleeding surfaces. In the manufacture of plasma derivatives from pooledhuman plasma, viruses become partitioned as part of the fractionationprocess. Because specific viruses partition with some fractions but notothers, in certain cases partitioning alone may be sufficient to clear aplasma derivative of a particular infective agent. However, from theAIDS epidemic, it is now known that while HIV may be effectively clearedfrom immunoglobulin, it can remain in antihemophilic factorconcentrates. It is, therefore, of great importance that all plasmafractions are assumed to be contaminated and that vigorous inactivationmethods be employed.

A number of viral inactivation strategies have been investigated and aredescribed in the prior art literature. For example viral inactivationmethods in blood products, include, but are not limited to dialysis,ultrafiltration, two-step vapor heating (cumulative), high temperatureand pressure sterilization, solvent detergents such as tri(n-butyl)phosphate (TNBP) or Tween 80, photochemicals such as psoralen analogs,pasteurization (heating), radiation exposure, and ultraviolet lighttreatment. Although virus inactivation by high heating or steam methodsare impractical for biologically active protein solutions, including thepresent fibrinogen solutions, nanofiltration is an optional treatmentwithout causing inactivation of the components, such as human fibrinogensolution, of the present invention before placing it into the sterilestorage container.

Viral inactivation methods that reduce infectivity by 5 logs shouldprovide assurance that a preparation is no longer infectious. Forinstance, the vapor heating process used in the production of Tisseel VHfibrin sealant has been shown to reduce viral titers by at least 6.4 logreduction units for each vapor heating step of the two step process,bringing the risk of contamination to negligible. Various washing stepscan be employed to remove stabilizing additives by methods known in theart. Methods known to effectively offer viral inactivation in to priorart fibrin sealant compositions may be used and will be equallyeffective for the instant FS products of the present invention.

Methods of Preparing and Using the FS Composition

The method of formulating the FS composition of the present inventionmay be performed in a number of ways, including, but not limited to thefollowing preparation techniques, which generally result in a wellformulated composition. The preparation is generally conducted at nomore than 22.5°-30° C. Initially, the fibrinogen and the activatorcomponents are formulated into sterile aqueous solutions at the desiredconcentration. The advantage of the present invention that has notpreviously been possible is that the aqueous solutions can then bestabily stored for days, weeks or months without significant change inactivity or ability to form the FS composition of the present inventionwhen combined. When the FS composition is needed, the fibrinogencomponent is neutralized and combined with the activator component,which may also contain CaCl₂, Factor XIII and other additives, dependingon the intended purpose of the FS. The components are combined in aratio, which is determined by the intended end-use of the composition.To improve the mixing of the molecules of the primary components, it isgenerally advantageous to agitate the composition either internally, orexternally, typically by stirring or shaking vigorously as described inthe following Examples, until a sol or gel forms.

In alternative embodiments, additives to enhance viscosity, the bond, orvisualization of the material may be added after the components arecombined. Other components, such as pH modifiers, stabilizers, proteaseinhibitors, surfactants, antioxidants, osmotic agents, preservatives andthe like may be added at this time, as well as components which do notaffect the FS per se, but are added for delivery to the patient ortissues in vitro or in vivo.

In a preferred embodiment of the present invention, no antimicrobialagent is added to the fibrinogen, rather sterility is preserved usingknown techniques. However, in an alternative embodiment, antimicrobialagents are added to the extent exemplified, to avoid microbialcontamination of the fibrinogen solution component over long termstorage. Any recognized, physiologically antimicrobial agent isacceptable for the purposes of the present invention, so long as theactivity of the fibrinogen solution is maintained throughout the lengthof the storage, spontaneous clotting is not induced, and the agent isnot contra-indicated for human use.

The instant fibrin sealant of the present invention, prepared fromstorage-stable fibrinogen components, such as human fibrinogen, may bethus used in any known manner in which such biologically-prepared,supplemented or unsupplemented tissue adhesives are used, e.g.,pharmacological or cosmetic uses, including for infusion purposes, suchas delivery of antibiotics, antineoplastics, anesthetics, and the like;as a soft tissue augmentor or soft tissue substitute in plasticreconstructive surgery; to attach skin grafts to a recipient sitewithout the use of sutures or with a reduced number of sutures, or as agrowth matrix for transplanted intact osteoblasts in bone repair andreconstruction. The FS can also be used for applications such asossicular reconstruction, nerve anastomosis or other situations whererepair by sutures is impossible or undesirable, or as a wound dressing.

The FS may be applied in a number of ways determined by the surgicalindication and technique for wound healing, coagulation andfibrinogenanemia; for inhibition of operative or post-operativesequelae; for coating prostheses; for dressable wound sealings and forsafe and sustained hemostasis, namely sealing fluid and/or air leakage,and the like in a patient. Certain preferred embodiments of theinvention provide methods of directly using the subject instant FScomposition for connecting tissues or organs, for example, withoutlimitation, to stop bleeding, heal wounds, seal a surgical wound, use invascular surgery, include providing hemostasis for stitch hole bleedingof distal coronary artery anastomoses, left ventricular suture lines,aortotomy and cannulation sites, diffuse epimyocardial bleeding asoccurs in reoperations, and oozing from venous bleeding sites. Thesubject invention is also useful for sealing Dacron and other graftsprior to insertion and for coating prostheses, stopping bleeding inspleens livers, and other parenchymatous organs, sealing tracheal andbronchial anastomoses and air leeks or lacerations of the lung, sealingbronchial stumps, bronchial fistulas and esophageal fistulas; forsutureless seamless healing, and embolization in vascular radiology ofintracerebral AVMs, liver AVMs, angiodysplasia of colon, esophagealvarices, sealing “pumping” gastrointestinal bleeders secondary to pepticulcers, and the like. The subject invention is further useful forproviding hemostasis in corneal transplants, nosebleeds,tonsillectomies, teeth extractions and other applications.

In each of the foregoing described applications, there is a break in thenormal tissue integrity of the patient. The location of the break or thesite of application of the FS is referred to herein collectively as a“wound site,” although it may not always be a wound per se. For example,an air leak is not necessarily a wound, nor is the addition of aprosthesis, but for the purpose of simplicity, they are collectivelyreferred to herein as a “wound” because each occurs at a break in thenormal tissue, and each is sealed or treated by application of the FScomposition of the present invention.

In the preferred practice of the present invention, the FS compositionis formulated and “instantly” converted, meaning concurrently withcontact with the wound site, or within 300 seconds, preferably within180-240 seconds, more preferably within 150-180 seconds, even morepreferably within 100-150 seconds, and most preferably in less than 100seconds the fibrin monomer forms and is converted to polymerized orpartially polymerized fibrin, and/or noncross-linked fibrin is convertedto cross-linked fibrin. In fact, the instant FS clot is typically formedin under 60 seconds, more often in under 30 seconds, and in the examplesprovided herein, the FS clot consistently formed in 8 to 30 seconds,most often in 9 to 12 seconds.

Thus, “instantly” or “concurrently” also refers to the fibrin-formingstep occurring upon activation of the storage-stable fibrinogencomponent, within 300 seconds, preferably within 180-240 seconds, morepreferably within 150-180 seconds, more preferably within 100-150seconds, more preferably in less than 100 seconds, more preferably inless than 60 seconds, more preferably in less than 30 seconds, mostpreferably in 8 to 30 seconds, and typically in 9 to 12 seconds.However, even in the longest of times, the FS of the present inventionis “instant” when compared to any prior art preparation because thefibrinogen is always ready-to-use in aqueous solution, as are the othercomponent(s) without time consuming and difficult measuring or separatemixing and preparation of such component(s) from lyophilized or frozenformulations or from fresh blood or plasma samples.

In one preferred embodiment, the fibrin forming step and the contactingstep at the wound site are “concurrent” meaning simultaneous, althoughpolymerization may take some additional period to complete within theabove-stated time ranges. However, the conversion step of the FScomposition components to fibrin occurs within 60 seconds of activationand/or contact, preferably within 30 seconds, more preferably within 15seconds, and yet more preferably within 10 seconds, most preferablywithin 0-10 seconds. Otherwise, the FS composition may flow away fromthe intended site.

Finally, instantly and concurrently can also mean that the conversionstep commences prior to the contacting step, albeit not so far inadvance of the contacting step that all of the fibrin monomer (resultingfrom activation of the storage-stable fibrinogen) has been polymerizedor converted to cross-linked fibrin. For example, this embodiment of theinvention could occur when the storage-stable fibrinogen component isactivated by exposure to thrombin or a thrombin-like enzyme in thepresence of calcium ions in a syringe-type device prior to applicationof the resulting combined composition to the wound site. If all of theresulting fibrin has been polymerized or converted to cross-linkedfibrin prior to the contacting step, the composition no longer retainsany fluidity and it can no longer form a satisfactory fibrin sealant,nor can it be any longer used for such purposes. Since it takes ideallytakes less than about 30 seconds for the storage-stable fibrinogencomponent to be converted to the FS fibrin composition, the conversionstep should not begin more than about 30 seconds, and preferably notmore than about 3-10 seconds prior to the contacting step, unless acomponent such as a chaotropic agent has been added to delay theconversion. This embodiment is preferred because it ensures that themaximum amount of the FS composition will polymerize at the desiredsite, while at the same time form an excellent fibrin clot. As a result,the FS composition of the present invention that has been prepared fromstorage-stable fibrinogen eliminates many possible variables in thepreparation of the sealant formulation, permitting instant applicationof the FS composition to the wound site under closely controlledconditions.

FS Delivery

The FS product of the present invention is conveniently formed by mixingat least two components just prior to use. The first component comprisesstorage-stable fibrinogen in ready-to-use aqueous solution, the secondis an activator component, typically thrombin or a thrombin-likeactivator and calcium ions. Factor XIII and/or other additive componentsmay also be included as described elsewhere in this specification. Ingeneral, the components are conveniently delivered using a two-syringesystem, wherein the syringes are joined by a syringe-to-syringeconnector having about a 1 mm or less diameter opening. Substantialuniformity can be achieved with simple, generally available equipment.

FS application in the prior art includes dual syringe devices, which mixthe fibrinogen and thrombin as they exit from a single port, typicallyusing a large needle to direct the flow onto the wound. However, suchdelivery systems are known to form clots within can cause needle andtube blockages. Known dual syringe systems are also awkward to fill andmanipulate, and if there is inadequate mixing of the fibrinogen andthrombin components, the resulting clots may lack strength orelasticity. Because many wound sites leak significant amounts of fluidat the site, improperly formed fibrin seals may become ineffective or beflushed away. These problems are, however, overcome by the presentinvention because the stable fibrinogen is stored in aqueous solution,in ready-to-use form without mixing, measuring or time delay, and it maybe directly stored in a pen-type syringe delivery device.

In the present invention, the components are mixed immediately prior topolymerization. The components may be formulated with concentrationsthat allow mixing the components in adequate volumes to simplify thefinal preparation of the adhesive, preferably the volumes aresubstantially equal. Conveniently, a dual-barrel syringe holder with adisposable mixing tip can be used. Alternatively, the two components canbe mixed using two syringes as described above, or the components may bedirectly applied to the wound site, whereupon mixing occurs at the site.Preferably, however, the components are thoroughly mixed before deliveryor as apart of the delivery process or form the FS composition at thetime of delivery to the site.

The double-barrel syringe can be Y-shaped, thereby permitting the mixingof the FS composition from the storage-stable components, and theactivation of the conversion step simultaneously with, or immediatelyprior to the contacting step. In the alternative, rather than a Y-shapeddouble-barrel syringe, a double-barrel syringe with two openings can beutilized. This permits the simultaneous contacting of the wound site andconversion to the FS polymer from the storage-stable components. In yetanother alternative embodiment, the storage-stable components of thedouble-barrel syringe can be sprayed onto the desired site (see Kram etal., Amer. Surgeon, 57:381 (1991)). In yet another alternativeembodiment, the storage-stable components are held in a single-barrelsyringe separated by a non-porous material that is punctured, broken,dissolved or simply removed to allow the mixing of components just priorto delivery of the then-converted FS.

In the alternative, the fibrinogen is easily drawn into the deliverydevice from a larger container using standard drug delivery techniquesused with medicaments delivered by syringe.

Fibrin Sealant Kits

Further provided in the present invention are kits for the ready-to-usedelivery of an instant FS composition comprising at least two vials. Onevial, which as previously noted is not glass, contains an aqueoussolution of storage-stable fibrinogen at a concentration suitable forforming FS when mixed with an activator solution, such as thrombin or athrombin-like composition, and a second vial contains an activatorsolution, which is preferably thrombin at a concentration suitable forforming FS when mixed with the contents of the storage-stable fibrinogenin the first vial. By “vial” is intended herein to include a barrel of asyringe and multiple vials include a multi-barrel syringe device.

The pH of the activator solution can be adjusted so that it willneutralize the fibrinogen component when the two are mixed, or aseparate vial of neutralizing buffer may be provided to neutralize thefibrinogen component before it is mixed with the activator component.

A source of calcium ions, such as CaCl₂, is added to and stored with thecontents of one of the at least two vials in an effective amount toensure fibrin polymerization, preferably it is combined with thethrombin activator component. In the alternative, the calcium (CaCl₂)component is supplied in an additional vial. Additional components, suchas a stabilizer and/or Factor XIII, and/or additives, such as a growthfactor, drug, antibiotic, and the like are supplied by one or moreadditional vials, or alternately such additional components are added toand stored with the contents of the at least two vials.

In a preferred embodiment of the invention the storage-stable fibrinogencomponent is supplied at a concentration ranging from about 75-115mg/ml, and thrombin is supplied at approximately 500 IU/ml. Whenpresent, CaCl₂ is supplied at approximately 40 mmol/liter. When present,a fibrinolysis inhibitor, such as aprotinin is supplied at approximately3000 KIU/ml. Additional components, when present, are supplied atsuitable concentrations as determined by the purpose for which they areadded. For example, an antimicrobial component intended to stabilize theFS components per se are supplied at low concentrations as exemplifiedherein, whereas an antimicrobial composition intended for slow-releasedelivery to the patient to whom the FS is applied, would be supplied ata much higher concentration. Such amounts or concentrations can bedetermined or would be known to those skilled in the medical formulationart. Similarly, such an individual would know whether two or morespecific components or additives can be combined and stored in a singlevial without contra-indication, or whether the same components oradditives will remain more independently active and storage-stable ifsupplied separately in individual vials.

The invention is further described by example. The examples, however,are provided for purposes of illustration to those skilled in the art,and are not intended to be limiting. Moreover, the examples are not tobe construed as limiting the scope of the appended claims. Thus, theinvention should in no way be construed as being limited to thefollowing examples, but rather, should be construed to encompass any andall variations which become evident as a result of the teaching providedherein.

EXAMPLES Example 1 FS Clotting Assay for Preparations Using FibrinogenStored in Aqueous Solution at Room Temperature, Neutral pH

To evaluate the ability to rapidly prepare FS compositions fromstorage-stable, ready-to-use aqueous solutions of fibrinogen, theclotting activity of fibrin sealants prepared using fibrinogen that hadbeen stored in aqueous solution for long periods of time were evaluated.

Bovine fibrinogen, bovine thrombin, buffer solutions, calcium chloride,sodium hydroxide and hydrochloric acid were purchased from SigmaChemical Company, St. Louis. Mo. Human fibrinogen was certified tocontain 53% protein (95% clottable) and 47% salts. Bovine fibrinogen wascertified to contain 61% protein (97% clottable) and 39% salts.

Standard research grade fibrinogen contains salts used in the isolationand purification process. This includes sodium citrate and sodiumchloride. Thus, a 40 mg/ml solution of fibrinogen contains, for example,54 mM sodium citrate and 419 mM sodium chloride in addition to thefibrinogen. Additionally, sodium azide (0.025%) was added to each sampleas an antimicrobial agent, although in a preferred embodiment of thepresent invention, no antimicrobial agent would be added to thefibrinogen, rather sterility would be preserved using known techniques.

The clotting assays were completed in the following manner in generalaccordance with Kasper, Proc. Symposium on Recent Advances in HemophiliaCare, Los Angeles. Calif. Apr. 13-15, 1989 (in Liss, N.Y., 1990).Aliquots (100 μl) of each fibrinogen sample were added to 4 mlpolypropylene test tubes. Each sample was neutralized (pH 7.0-7.5) using0.1 M sodium hydroxide, 0.2 M histidine buffer (pH 6.0) or 0.1 Mhydrochloric acid (determined in preliminary studies using largervolumes). Thrombin was prepared as 200 units/ml with 1 M calciumchloride (3-6 mM excess of calcium over sodium citrate in fibrinogenpreparations). The thrombin preparation was then diluted with 0.1 Mhistidine buffer (pH 7.2) to a final thrombin concentration of 100units/ml (2.5 units of thrombin per mg of fibrinogen). All samples wereassayed at room temperature (23±2° C.).

Clotting was measured by timing the reaction that occurred when 100 μlof thrombin was added to the fibrinogen sample (100 μl), and the mixturewas vigorously mixed. Times were recorded when the solution turned to aviscous gel (a drastic slowing of the liquid being mixed) and to a solidclot (the point at which all liquid ceased movement upon mixing). Thetime to solid clot formation was often twice the time of gel formation.

In accordance with the described procedure, a manual clotting assay wasperformed at 25° C. by neutralizing the stored fibrinogen solutions; andadding thrombin (125 units/mg fibrinogen), and 3-5 mM excess CaCl₂ overcitrate in the fibrinogen solution. The preparation was mixedvigorously, and the time required for a clot to form was measured asdescribed above.

Clotting results using bovine fibrinogen in histidine buffer at pH 7.24,stored in aqueous solution at room temperature (˜23° C.) were asfollows: using fibrinogen stored for 3 days a clot formed within 9seconds, stored for 36 days a clot formed within 10 seconds, and storedfor 72 days (more than 10 weeks) a clot formed within 9.5 seconds. Thus,the clotting assay results are consistent regarding the time needed toform a FS clot in a preparation prepared from fibrinogen stored at roomtemperature for long periods of time in ready-to-use aqueous solutions(at neutral pH).

Example 2 FS Clotting Assays Using Fibrinogen Solutions Stored at TwoTemperatures, and a Range of pH Values

To evaluate the ability to rapidly prepare FS compositions fromstorage-stable, ready-to-use aqueous solutions of fibrinogen, theclotting activity was evaluated of fibrin sealants prepared usingfibrinogen that had been stored in aqueous solution for long periods oftime over a range of pH values (pH 6.50 to pH 9.87), at room temperature(˜23° C.) and refrigerated (4° C.). Duplicate solutions of fibrinogenwere evaluated in clotting assays as described in the stability study inExample 1.

Clotting results are shown in Table 1 bovine fibrinogen (39 mgprotein/ml) and in Table 2 for human fibrinogen (40 mg protein/ml),respectively prepared for storage in one of the following 0.1 M buffers:histidine, pH 6.0 or 7.2; Tris pH 8.16. TABLE 1 Clotting times forbovine fibrinogen, stored at 23″ C. and 4″ C. Age in Temp. in ClottingTime (in seconds) Days ° C. pH 6.5 pH 7.36 pH 8.2 pH 9.04 pH 9.87 4 2312 13 15 12 210 4 10 9 15 10 Clotted 7 23 10 10 11 11 240 4 11 10 10 10Clotted 22 23 9 10 10 >300 >300 4 Partial clot Partial clot ClottedClotted Clotted 97 23 10 100 >300 >300 Clotted 4 Clotted Clotted ClottedClotted ClottedNT = not tested.“Clotted” refers to spontaneous clotting, absent addition of thrombin.

TABLE 2 Clotting times for human fibrinogen stored at 23° C. and 4° C..Age in Temp. in Clotting Time (in seconds) Days ° C. pH 6.32 pH 7.13 pH8.04 pH 8.79 pH 9.43 4 23 10 10 11 12 120 4 10 10 9 10 Clotted 7 23 1010 9 11 240 4 10 9 8 9 12 22 23 10 8 10 >300 >300 4 10 8 9 NT NT 97 2330 >300 >300 >300 >300 4 18 10 10 11 >300 149 23 NT >300 >300 NT NT 4 15135 30 >300 >300NT = not tested.“Clotted” refers to spontaneous clotting, absent addition of thrombin.

Each and every patent, patent application and publication that is citedin the foregoing specification is herein incorporated by reference inits entirety.

While the foregoing specification has been described with regard tocertain preferred embodiments, and many details have been set forth forthe purpose of illustration, it will be apparent to those skilled in theart that the invention may be subject to various modifications andadditional embodiments, and that certain of the details described hereincan be varied considerably without departing from the spirit and scopeof the invention. Such modifications, equivalent variations andadditional embodiments are also intended to fall within the scope of theappended claims.

1. A ready-to-use, instantly available fibrin sealant (FS) compositionprepared from a storage stable, aqueous fibrinogen solution componentand an activated thrombin or thrombin-like component. 2-18. (canceled)